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27/2/2015 Ciencia - Three constituents with biological activity from Coccoloba uvifera seeds

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CienciaPrint version ISSN 1315-2076

Cienciavol.16no.1MaracaiboMar.2008

Three constituents with biological activity from Coccoloba uviferaseeds

Shailili Moreno Morales 1*, Oscar Crescente Vallejo 2, WilliamHenrquez Guzmn 2, Gustavo Liendo Polanco 2 and Hernando

Herrera Mata 2

1 Laboratorio de Fitoqumica Biodirigida, Departamento de Qumica y

procesos. Universidad Simn Bolvar (USB), Apartado 89000, Caracas 1080-

A, Venezuela.

2 Laboratorio de Productos Naturales, Departamento de Qumica, Escuela de

Ciencias. Universidad de Oriente (UDO), Ncleo de Sucre, Cuman 6101,

Venezuela.

* Autor para la correspondencia. Telf.: 0414-8405948. E-mail:[email protected].

Abstract

Uvero de playa (Coccoloba uvifera Jacq.) seeds were extracted with methanol and the antibacterial, antifungal,toxic and phototoxic activities were determined together with their phytochemical analysis. The extract was activeagainst Salmonella typhimurium and Staphylococcus aureus, showing the presence of flavonoids, saponins,polyphenols and tannins. The partition with EtOAc of the MeOH extract gave a brown precipitate that inhibited thegrowth of Escherichia coli and Pseudomonas aeruginosa, and showed antifungal activity against Candida albicansFusarium oxysporum and F. decencellulare. This solid was fractionated by means of vacuum columns (low pressurecolumns) chromatographies, molecular exclusion and bidimensional paper chromatographies obtaining a tanniccompound, an organic acid and a benzopyran from the bioactive fractions. These compounds were identified bymeans of IR, GC/MS and NMR (1H and 13C) as: gallic acid, hexenedioic acid and 1,3,4,6,7,8hexahydro-4,6,6,8,8,8-hexamethylcyclopenta-2-benzopyran.

Key words: Benzopyran; Coccoloba uvifera; gallic acid; hexenedioic acid; Polygonaceae.

Tres constituyentes con actividad biolgica de las semillas de Coccoloba uvifera

Resumen

Las semillas del uvero de playa (Coccoloba uvifera Jacq.) fueron extradas con MeOH y se les determin suactividad antibacteriana, antifngica, txica y fototxica; adems se les realiz un anlisis fitoqumico. El extractofue activo contra Salmonella typhimurium y Staphylococcus aureus; tambin mostr reaccin positiva en laspruebas de flavonoides, saponinas, polifenoles y taninos. Se particion el extracto metanlico con AcOEt,obtenindose un precipitado que inhibe el crecimiento de Escherichia coli y Pseudomonas aeruginosa, y mostractividad antifngica contra Candida albicans, Fusarium oxysporum y F. decencellulare. Este slido fue fraccionadopor columnas cromatogrficas de: vaco, exclusin molecular y bidimensional de papel, obteniendo un compuestotnico, un cido orgnico y un benzopirano de las fracciones bioactivas. Estos compuestos fueron identificados pormedio de IR, CG/EM y RMN (1H y 13C) como: cido glico, cido hexenedioico y 1,3,4,6,7,8-hexahidro-4,6,6,8,8,8-hexametilciclopenta-2-benzopirano.



Palabras clave: cido glico; cido hexenedioico; benzopirano; Coccoloba uvifera; Polygonaceae.

Recibido: 26-06-07 Aceptado: 22-05-08

Introduction

In the Polygonaceae family we can find species of the Coccolobas gender, many of them are used to treat somediseases. Some of these plants have been analyzed for their biological activity and their chemical constitution. TheCoccoloba barbadensis dry leaves are used in Mexico for the treatment of kidneys illness in adults (1), while inIndia the Coccoloba diversifolia bark have been boiled in order to stop the diarrhea (2). In 1996 Nicaraguanscientists evaluated the biological activity of some coccoloba extracts, the aqueous extract of the C. cozumelensishad antibacterial activity against Staphylococcus aureus, Bacillus punullus y Pseudomonas aeruginosa, the C.pubescens ethanol extract exhibit an antimalarian activity (3). Concerning to the chemical therapeutic agentsisolation, it has been obtained a successful substance like gallic acid and benzoic acid from Coccoloba dagandicena(4), also it has been isolated betulinic acid, lupeol and sitosterol obtained from Coccoloba excoriata (5).

The Coccoloba uvifera Jacq. is another vegetable species that belongs to this family of plants. This species isoriginal from the Antilles, the Bahamas and South American tropics; it could be found frequently in Venezuelancoasts by the common name sea grape. This plant has been used whit pharmaceutic purposes, the roots andbarks boiled in hot water are used in popular medicine against diarrhea and dysentery. Some studies show that thebark contain tannins used to dye (6), and other investigations show that it is the source of quine, resin of medicalinterest that appears in the wound of the trunk. Coccoloba uvifera Jacq. (Polygonaceae) is a very commonmedicinal plant on Venezuelan coasts. Previous studies have reported the presence of some secondary metabolitesin its leaves and bark (7), but neither the chemical composition of its seeds or the biological activity of its extractshas been studied.

Coccoloba uvifera Jacq. effectivity in the treatments indicated above what is a potential source of secondarymetabolism with significant biological activity, which could be present either in the leaves, the bark and fruit seeds.This species have not been studied yet, in spite of their medical uses. In addition we dont have any information orreports about secondary metabolites in this part of the plant, making of great interest that is why it has beenselected the Coccoloba uvifera in order to isolate the responsible components of the biological activity.

Materials and methods

Plant material

Seeds from C. uvifera were collected in July 2001 in the gardens of the Universidad de Oriente in Cuman, State ofSucre, Venezuela. The plant was identified by Prof. Luis Jose Cumana. A voucher specimen (No. 01072001) waskept in the IRBR Herbarium at the same university.

Extract preparation

Plant material (93.5 g) was dehydrated in the shade at room temperature. It was then grinded in an electric mill,and the resulting powder was weighed and extracted successively with MeOH until exhaustion. The MeOH seedextract (yield: 21.6%) was dissolved in water and kept in the refrigerator for 24 h at 4C, and a resulting brownsolid was separated by filtration. The remaining aqueous solution was continuously and exhaustively extracted withAcOEt and the organic layer was rotaevaporated after drying over MgSO4 (the brown extract was named ASE).Chemical tests performed on the former extract gave positive results for polyphenols and catechinic and gallictannins (8).

Polyphenols and tannis test

Phenolic compounds are detected by the brown coloration that take place in presence from a solution of FeCl3 to1%. For that the total extract is evaporated to dryness, dissolved in H2O and filtrated before the reaction withferric chloride. If the phenols are present, they produce a brown coloration. If the raw contains tannis, a precipitateis produce with 1% Jello in 1% NaCl sln.

Catechinic and gallic tannins test

The sample is dissolved in MeOH-H2O 1:1, 1mL of this solution is added to 8mL of Stiasny reagent (twiceformaldehyde volumens to 30% + a volume of concentrated HCl) it warms to boil for 30min, the presence ofcatechinic tannis is evidenced by appearance of a brown precipitate, it is filtrated and the solution is saturated withsodium acetate, them are added some drops of 2% ferric chloride. A blue coloration indicates the presence of gallictannis.

Biological activity



All extracts and fractions were evaluated by means of antibacterial (9), antifungal (10), toxic (11) and phototoxic(12) bioassays. Antibacterial and phototoxic tests were realized using the Gram-positive bacteria strainsStaphylococcus aureus Rosenbach (ATCC 6538P) and Bacillus cereus Frank. Frank. (ATCC 9634); while the Gram-negative ones were: Escherichia coli T. Esch. (ATCC 0389), Pseudomonas aeruginosa Schroeter (ATCC 25416) andSalmonella typhimurium D. E. Salmon (ATCC 14028). In the toxicity test brine shrimp Artemia salina Linn.,Crustacea, Anostraca was used. Two opportunistic fungi (Candida albicans Langenbeck and Trichosporum sp.Elmer) and two phytopathogenic fungi (Fusarium oxysporum Killiam Maire and F. decencellulare Bender) were usedfor the antifungal assay.

The biological activity was used to biodirection the whole isolation process.

Isolation

Column chromatography was done on a silica gel Aldrich 2070 mesh 60 (123.5g) starting with hexane followedby dichloromethane, acetone, methanol, methanol/water (9:1) and methanol/acetic acid (9:1). Molecular exclusionchromatography was performed on Sephadex LH 20, 25-100 m starting with AcOEt (100%), followed by AcOEt-MeOH (1:1), MeOH (100%) and MeOH-H2O (8:2). For vacuum chromatography Sigmacell cellulose type 50 S-5504was employed with dichloromethane, acetone and methanol using a Cole-Parmer pump, model 7049-00.

IR spectra were recorded on a Perkin Elmer FTIR 16 PC spectrophotometer using KBr tablets, 24 scans and a 2 cm1 resolution. The GC-MS was performed with a Hewlett Packard model 5890, series II, gas chromatograph coupledto a EI (70eV) mass spectrometer, Hewlett-Packard, model 5971 A. A methyl silicone column DB-5 (25 m) wasused, with a 280C injector temperature. The initial and final temperatures were 70C and 310C, respectively(rate of heating 6C/min) with helium as carrier gas. The 1H, 13C NMR spectra were recorded on Jeol 400 MHzspectrometer using deuterated methanol, acetone and water like reference standard.

Gallic acid (A). IR bands (KBr): 3500 3000, 1700, 1600, 760 cm-1; EIMS m/z: 170 (M+), 152, 125 (base peak),110; 1H NMR (400 MHz, MeOH d4): d 6.9 7.4 (H aromatic and phenolic); 13C - NMR (400 MHz, MeOH d4):d 170.4 (C = O), 146.4 (C 3 and C 5), 139.6 (C 4), 121.9 (C 1), 109.9 (C 2 and C 6).

Hexenedioic acid (B). EIMS m/z: 144 (M+), 126, 108, 57 (base peak).

1,3,4,6,7,8-hexahydro-4,6,6,7,8,8-hexamethylcyclopenta-2-benzopyran (C). EIMS m/z: 258 (M+), 243 (basepeak), 227, 213, 198, 185, 171, 158, 143, 128, 113, 86. 1HNMR (400 MHz, DMSOd6): d 7.2 (H5), 4.6 (H4), 4,4(H7), 3.9 (H1), 3.6 (H3), 1.6 (H4), 1.2 (H6), 0.8 (H7); 13C - NMR (400 MHz, DMSOd6): d 128.9 and 131.0 (Caromatic), 66.9 (C7), 63.1 (C3), 30 (C6 and C-8), ~ 23 (C6, C8 and C4), 14.1 (C7).

Results and Discussion

The MeOH extract from the seeds (MSE) of Coccoloba uvifera gave positive results for the catechinic tannins testand an antibacterial effect against Salmonella typhimurium and Staphylococcus aureus was observed with halosinhibition diameters of 15 mm and 17 mm respectively, this test was carried out at 40mg/mL of concentration.However, the extract did not show any antifungal, toxic or phototoxic activities. MSE was partitioned with AcOEtgiving a brownish organic extract (ASE) that gave positive results for the tests for polyphenols and tannins andshowed an antibiotic effect against Escherichia coli, Pseudomonas aeruginosa, Candida albicans, Fusariumoxysporum and F. decencellulare. ASE (5.70g) was chromatographed by gravity giving 25 fractions. Fractions 7 to14 showed positive results for polyphenols and catechinic tannins, except for fraction 10 that showed the presenceof gallic tannins. Fractions 6 to 14 showed antibacterial activity (Table 1). The solvents also used like blanks, didntshow biological activity.

Table 1. Antibacterial activities from ASE fractions expressed in mm.

Microorganism F6 F7 F8 F9 F10 F11 F12 F13 F14

Escherichia coli 12 12 14 13 12

Pseudomonasaeruginosa

16 20 16 12

Salmonella 15 12 20 20 12



typhimurium

Staphylococcus aureus 12 17 13 24 22 12 11 11

Bacillus cereus 11 11 12 12 20 17 11

F: Fraction.

Fractions 9, 10 and 11 were rechromatographed by column vacuum and molecular exclusion chromatographies. Allsubfractions were studied by IR, GC/MS and 1H and 13C NMR identifying gallic acid (A) from F10-5-3; hexenedioicacid (B) from F11-4-5-2 and 1,3,4,6,7,8 hexahydro-4,6,6,8,8,8-hexamethylcyclopenta-2-benzopyran (C) from thesame subfraction (Figure 1).

Figure 1. A: Gallic acid; B: Hexenedioic acid; C: 1,3,4,6,7,8- hexahydro-4,6,6,7,8,8- hexamethylcyclopenta- 2- benzopyran.

Gallic Acid. The IR spectrum showed absorption bands at 3000 3500 (vR OH), 1700 (vC= O), ~ 1600 (vC= Caromatic), ~ 760 (dC H aromatic) cm-1. The EIMS showed an ion peak at m/z 152 that corresponds to the loss ofH2O. The intense peak at m/z 125 represents the loss of the carboxylic group and formation the C6H5O3].+.The 1H NMR spectrum showed resonances between d 6.9 and 7.4 ppm, that were attributed to aromatic and phenolicprotons. The 13C NMR spectra of commercial gallic acid and the obtained acid in our work, showed identicalabsorptions (Table 2). This acid has previously been isolated from Coccoloba dugandiana Fernandez(Polygonaceae) (4).

Table 2. Chemical shifts (ppm) for the different carbons of F10 5 3 and commercial gallic acid.

Carbon d [F10-5-3] d [Gallic acid]

C1 121.9 121

C2 and C6 109.9 110

C3 and C5 146.4 146

C4 139. 6 140



C = O 170.4 168 - 170

Hexenedioic Acid. Its EIMS showed a peak at m/z 144 corresponding to the molecular mass of the acid, and a peakat m/z 126 formed by the loss of H2O producing [C6H6O3].+.

1, 3, 4, 6, 7, 8Hexahydro-4, 6, 6, 7, 8, 8Hexamethylcyclopenta2Benzopyran. Its EIMS showed a peak at m/z258 corresponding to C18H26O, m/z 243 corresponding to the ion formed by the loss of a methyl group. The 1H NMR spectrum exhibited signals assigned to protons in the methyl terminal groups [d 0.8 (H7), 1.2 (H6) and 1.6(H4) ppm]. At d ~3.6 and ~3.9 ppm two signals appeared which were attributed to the two methylene groupsadjacent to the oxygen atom, whilst two multiplets at 4.4 and 4.6 ppm correspond to the methyne protons (H7and ~4,6 (H4) ppm. Aromatic protons H5 and H9 appear as a singlet at 7.2 ppm. The 13C NMR spectrum showedthe methyl group C7 at 14.1 ppm, whilst the methyl groups at C6, C8 and C4 appear around 23 ppm. Signalsaround 30 ppm correspond to the quaternary carbons, aromatic carbons appear at 128.9 and 131.0 ppm whilst twosignals at 63.1 and 66.9 ppm correspond to C3 and C7, respectively. In a previous study (13) benzopyraniccompounds with organic acids and tannins were isolated from propolis.

Conclusions

The presence of tannis in Coccoloba uvifera seeds was demonstrated.

The seeds methanolic extract inhibit the growth of S. aureus and S. typhimurium the fractions and sub fractions ofCoccoloba uvifera seeds also show antibacterial activity, these may have been considerate potential fountain ofuseful compounds for the combat of pathogens effect of these bacteria.

The seeds methanolic extract also contain compounds with antifungal activity against C. albicans, F. oxysporumand F. decencellulare.

In the sub fractions of Coccoloba uvifera seeds were identified: Gallic acid, hexenedioic acid and 1,3,4,6,7,8-hexahydro -4,6,6,8,8,8-hexamethylcyclopenta-2-benzopyran. These have antibacterial and antifungal activity.

References

1. ZAMORA M., POLA C. Ethnopharmacology 35 (3): 229-257, 1992. [ Links ]

2. AYENSU E. Unpublished results. [ Links ]

3. COE F., ANDERSON G. Ethnopharmacology 53: 29-50, 1996. [ Links ]

4. LI X., ELSOHLY H., NIMROD A., CLARK A. Planta Mdica 65 (18): 780, 1999. [ Links ]

5. COMPAGNONE R., CASTILLO A., DELLC F. Rev Colomb Quim 24 (2): 65-68, 1995. [ Links ]

6. LE B., RENARD M. 1997. Fruits from America. http://homepage1.nifty.com/yinz/Polygo.htm. Consulta: junio,2000. [ Links ]

7. KAWASAKI M., KANOMATA T., YOSHITMA K. Bo. Mag 99: 63-74, 1986. [ Links ]

8. MARCANO D., HASEGAWA M. Fitoqumica Orgnica. Universidad Central de Venezuela, Caracas (Venezuela),pp. 588, 2002. [ Links ]

9. BAUER A., KIRBY W., SHERRIS I., TURK M. Am J Clin Pathol 45: 493-496, 1996. [ Links ]

10. MADUBUNYI I. Int J Pharm 33: 232-237, 1995. [ Links ]

11. MEYER B., FERRIGNI N., PUTNAM J. JACOBSEN L., NICHOLS D., MCLAUGHLING J. Planta Mdica 45: 31-34,1982. [ Links ]

12. DANIELS F. J Inv Dermatol 44: 259-263, 1965. [ Links ]

13. BANSKOTA A., TEZUCA Y., ADYANA I., ISHII E., MIDORIKAWA K., MATSUSHIGE K., KADOTA S.Phytomedicine 8: 16-23, 2001. [ Links ]

2015 La Universidad del Zulia



: Facultad Experimental de Ciencias, La Universidad del Zulia, Maracaibo 4001-A, Venezuela. Telfono: +58261 7597719, 7598098. Tel-Fax: +58 261 7598099

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